The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target topoisomerase I (topo I) as a treatment for HIV-infection. In a preferred embodiment, the present invention relates to therapeutic protocols designed to interfere with the interaction of topo I with the HIV proteins gag and reverse transcriptase (RT), as a treatment for HIV-infection. The present invention still further relates to transgenic animals which express human topo I, in particular transgenic mice, and their use as a system to screen agents for their ability to interfere with the HIV life cycle.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. In humans, HIV replication occurs prominently in CD4+ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
The HIV viral particle comprises a viral core, composed in part of capsid proteins, together with the viral RNA genome and those enzymes required for early replicative events. Myristylated Gag protein forms an outer shell around the viral core, which is, in turn, surrounded by a lipid membrane envelope derived from the infected cell membrane. The HIV envelope surface glycoproteins are synthesized as a single 160 Kd precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD4+ cells because a CD4 cell surface protein (CD4) acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gp120 binding the cellular CD4 receptor molecules (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348), explaining HIV""s tropism for CD4+ cells, while gp41 anchors the envelope glycoprotein complex in the viral membrane. While these virus:cell interactions are necessary for infection, there is evidence that additional virus:cell interactions are also required.
Purified HIV virions have been reported to have topoisomerase activity (Priel et al., 1990, EMBO J. 9:4167-4172). Topoisomerase I is an enzyme that modifies the topological state of the DNA by breakage and rejoining of single DNA strands and relaxing both positive and negative supercoils. In addition to playing a role in normal cell function, topo I of cellular origin has also been shown to be involved in the replication of a number of DNA viruses, where it has been associated with DNA replication, transcription, and integration (Wang et al. 1990, J. Virol. 64:691-699; Shaack et al., 1990, Nucleic Acids Research 18:1499-1508; Kawanishi 1993, J. Gen. Virology 74:2263-2268). It has been suggested that the topo I activity associated with HIV virions is viral in origin in that it differs from cellular topo I in its requirements for optimal enzyme activity (Priel et al., 1990, EMBO J. 9:4167-4172).
HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H. et al., 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition, the drugs often cause toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith, D. H. et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al., WO 92/22,654; Shafferman, A., WO 91/09,872; Formoso, C. et al., WO 90/07,119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.
The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target topo I for the treatment of HIV infection. The invention relates to therapeutic modalities and pharmaceutical compositions for the treatment of HIV-infection using human topo I and its interaction with HIV gag and RT as a target for intervention.
The present invention relates to animal cell lines expressing human topo I, in particular mouse cell lines, and their use as a system to study the HIV life cycle and screen agents for their ability to interfere with the HIV life cycle. The present invention also relates to human topo I transgenic animals, in particular mice, and their use as a system to study the HIV life cycle and to screen agents for their ability to interfere with the HIV life cycle.
The invention is based, in part, on the Applicants"" surprising discoveries that (1) human topo I interacts with and is activated by HIV gag in a species specific manner; (2) the interaction between human topo I and gag is required to enhance HIV RT activity; and (3) the interactions between human topo I and HIV gag and RT are required to support HIV replication. This model is based on the Applicants"" observation that murine cells expressing human CD4 are not able to support HIV replication. However, murine cells support HIV replication. The expression of human topo I was also shown to enhance the activity of HIV RT in murine cells.
That human topo I interacts with and is activated by HIV gag, and that this complex is required for activation of HIV RT is further supported by the working examples described infra which demonstrate (1) that gag proteins activate cellular topo I and immunoprecipitated-gag proteins induce topo I activity in a species specific manner; (2) mouse cells expressing both human CD4 and topo I infected with HIV effectively reverse transcribe the HIV RNA genome; and (3) the topo I inhibitor, TAN134A, which attacks the topo I site directly, inhibits HIV RT activity in murine cells.
The invention further relates to a murine model for HIV replication, in which transgenic mice expressing both the human CD4 cell surface protein and human topo I are able to support HIV-1 replication. The present invention also encompasses a murine model for HIV replication, in which transgenic mice express human topo I and a HIV pseudovirus is used to infect the animals. The HIV pseudovirus may contain an envelope protein from a virus with a natural tropism for murine cells, such as the murine leukemia virus, which bypasses internalization of the HIV virus by the murine cells. These transgenic mice have utility to screen for other host cellular components required to support the HIV life cycle (i.e., entry, replication and assembly), in addition to screen for drugs and compounds which may have anti-HIV activity.
The invention relates to various modalities of treatment for HIV infection based on the proposed model. The invention further relates to the use of the murine HIV model system for screening test compounds, such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to interfere with the interaction between human topo I and HIV gag and RT.
The present invention further relates to the use of such identified inhibitors in pharmaceutical compositions designed to inhibit human topo I and/or the interaction between human topo I and HIV gag and/or HIV RT for the treatment and/or prevention of HIV infection. The present invention further encompasses the preparation of such pharmaceutical compositions for the treatment and/or prevention of HIV infection.
The invention also encompasses combinations of a topoisomerase I inhibitor with a least one other antiviral having a different site of action than the viral replication inhibitor. Such a combination provides an improved therapy based on the dual action of these therapeutics whether the combination is synergistic or additive.
As used herein, the term xe2x80x9ctopoisomerase Ixe2x80x9d or xe2x80x9ctopo Ixe2x80x9d refers to the protein topoisomerase I which has the activity of modifying the topological state of DNA, and any derivative of topo I thereof, or fragments or peptides having an amino acid sequence corresponding to topo I.
As used herein, the term xe2x80x9cHIV pseudovirusxe2x80x9d refers to a HIV virus that expresses a coat proten or an envelope protein which renders the virus capable of infecting a mouse cell. An example of such a protein is the envelope protein of the murine leukemia virus.
As used herein, the term xe2x80x9cto targetxe2x80x9d means to inhibit, block, or prevent gene expression, enzymatic activity, or interaction with other cellular or viral factors.
As used herein, the term xe2x80x9ctreating or preventing HIV infectionxe2x80x9d means to inhibit the replication of the HIV virus, to inhibit HIV transmission, or to prevent HIV from establishing itself in its host, and to ameliorate or alleviate the symptoms of the disease caused by HIV infection. The treatment is considered therapeutic if there is a reduction in viral load, decrease in mortality and/or morbidity.
As used herein, the term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient, is chemically inert and is not toxic to the patient to whom it is administered.
The term xe2x80x9ctherapeutic agentxe2x80x9d refers to any molecule compound or treatment, preferably an antiviral, that assists in the treatment of a viral infection or the diseases caused thereby.